Tsh immunoassays and processes for performing tsh immunoassays in the presence of endogenous contaminants in restricted wash formats

ABSTRACT

The invention relates to low wash Thyroid Stimulating Hormone (TSH) immunoassays using an ELISA sandwich assay having limited or no wash step between the antigen capture, detection antibody addition and substrate introduction steps. This invention exhibits low cross reactivity with biologically similar interfering cross reacting species, such as Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and Chorionic Gonadotropin (CG).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/306,536, filed Nov. 29, 2011, which claims priority to U.S.Provisional Application No. 61/491,088, filed on May 27, 2011, theentire contents of which are hereby incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates generally to methods of performing an immunoassayin systems having minimal washing capabilities. In particular, thisinvention relates to methods of performing a Thyroid Stimulating Hormone(TSH) assay in the presence of its related contaminating(cross-reacting) endocrine glycoprotein hormone analogues. The inventionalso relates to processes for selecting antibodies with the bestperformance characteristics for performing TSH assays. These assays alsobenefit from the inclusion of scavenging reagents that furtherameliorate the effect of interfering substances such as the endocrineglycoprotein hormone analogues.

BACKGROUND OF THE INVENTION

Immunoassays, first developed in 1959, have become useful sensitivediagnostic tools for human and veterinary health, and environmentaltesting (Wu, 2006, Clinica Chimica Acta, 369:119). Their application hasbecome ubiquitous and well known in the art.

There are several approaches to perform immunoassays, and this inventionfocuses on non-competitive or sandwich immunoassays. Immunoassays canalso be categorized as heterogeneous or homogeneous. Homogeneous assayshave simpler assay characteristics, but have been limited to highconcentration drugs (Engel & Khanna, 1992, Journal of ImmunologicalMethods, 150:99). Heterogeneous assays require the separation of boundmaterial from unbound material and are the focus of the presentinvention.

Sandwich immunoassays are typically performed as two step assaysinvolving first the introduction of an antigen-containing sample to acapture antibody or antibodies covalently attached to a solid support.This is followed by washing away non-specific sample components, leavingthe antigen of interest bound to the solid support antibody. In a secondstep, a detection or signal antibody is introduced into the assay,followed by another wash step. Lastly, the detection substrate is addedto the assay to quantify the antigen concentration. Two stepimmunoassays with associated wash steps are generally preferred as theyreduce background signal permitting highly sensitive detection.

A “one step” sandwich immunoassay may be performed by introducing thedetection antibody and antigen containing sample together to a captureantibody or antibodies covalently attached to a solid support. Theresulting assay is washed to remove unbound reagents. Lastly, thedetection substrate is added to the assay to quantify the antigenconcentration.

The need exists for one step assays with limited wash fluid volume, asthey are simpler and require fewer steps and fewer associated hardwarecomplexities in order to operate, particularly in point-of-careapplications. For point-of-care devices, the reducing or eliminating theneed for a wash step reduces device cost and assay time, hardware costand complexity, and disposable device size, which in turn is of benefitto reducing waste in the environment, as well as its cost.

To date, one step immunoassays with limited or no wash steps have notbeen used for antigens where the presence of endogenous related antigenscreate high backgrounds that confound detection results. This isparticularly true when the endogenous antigens are found at high molarconcentrations in excess of the antigen of interest, which is common forsome disease conditions.

An example of a problematic antigen is thyroid stimulating hormone(TSH), also known as thyrotropin, which is typically present incombination with the related endocrine glycoprotein hormones chorionicgonadotropin (CG), luteinizing hormone (LH), and follicle stimulatinghormone (FSH). These four related hormones have an identical alphasubunit and a highly similar beta subunit (Vassart, 2004, Trends inBiochemical Sciences, 29(3):119). Consequently, antibodies against thealpha subunit do not discriminate between these four hormones (Wada,1982, Clinical Chemistry, 28(9):1862). It is also difficult to identifybeta-specific antibodies that can discriminate amongst the four relatedhormones, particularly in the presence of very high concentrations ofcontaminating hormones, as these hormones have very similar primarysequences (Cornell, 1973, The Journal of Biological Chemistry,248(12):4327). Further, it is difficult to identify unique (antigenic)epitopes on TSH and to obtain antibodies that recognize these uniqueepitopes. For sandwich assays, it is also important to employ captureand signal antibodies that do not overlap having the appropriatespecificity characteristics, further limiting the choice of antibodies.

Wash steps in sandwich immunoassays help to reduce the concentration ofcontaminating hormones in the reaction, which reduces the signalassociated with the contaminating species. Therefore, one step sandwichimmunoassays with no wash step or limited wash capabilities have beenunable to specifically detect TSH in the presence of the relatedcontaminating hormone molecules, especially when the contaminants arepresent at very high concentrations. See, e.g., US20080311676, whichdescribes the importance of using a wash step in an immunoassay toreduce the concentration of cross-reacting species. Attempts to addressthese problems can be found in Hashida et al. 1986, Analytical Letters,19, 1121-36; Soos et al., 1984, Journal of Immunological Methods, 73,237; and Lode et al., 2003 Clinical Biochemistry, 36, 121.

EP 173973 describes a method for the determination of TSH, in which ananti-β-subunit TSH monoclonal antibody having a specific associationconstant value is used. Related EP 212522 describes an assaycharacterized in that TSH-β subunit specific monoclonal antibodiesrecognize different epitopes. This method seeks to reduce inhibition bythe presence of other glycoprotein hormones such as LH, CG and FSH.

The need exists for one step sandwich immunoassays, particularly thoseused for point-of-care assays, having reduced or eliminated washcapabilities, and in particular to one step sandwich immunoassays fordetecting TSH. In addition, the need exists for identifying antibodyreagents capable of performing such immunoassays.

SUMMARY OF THE INVENTION

The present invention relates to devices, immunoassays and methods fordetecting thyroid stimulating hormone (TSH) in a fluid sample, whileminimizing interference caused by the highly related endogenousendocrine glycoprotein hormones, Follicle Stimulating Hormone (FSH),Luteinizing Hormone (LH), and Chorionic gonadotropin (CG).

In one embodiment, the invention is to a thyroid stimulating hormone(TSH) sandwich immunoassay, comprising at least two epitope-compatibleantibodies comprising at least one capture antibody and at least onesignal antibody, wherein the dissociation constant (Kd) of bothantibodies for TSH is less than or about 1 nM, e.g., no greater than 0.5nM or no greater than 0.15 nM, and wherein the capture antibody Kd for:follicle stimulating hormone (FSH) is greater than 1000 nM, greater than2500 nM or greater than 3000 nM; luteinizing hormone (LH) is greaterthan 500 nM, greater than 1000 nM or greater than 3000 nM; and chorionicgonadotropin (CG) is greater than 200 nM, greater than 500 nM or greaterthan 2500 nM, and wherein the immunoassay uses a single wash step.

In another embodiment, the invention is to a method for performing a TSHimmunoassay, the method comprising the steps of: (a) inserting a fluidsample into a device comprising an immunosensor in a conduit, saidimmunosensor having a capture antibody to TSH; (b) dissolving a signalantibody into said fluid sample; (c) forming a sandwich complex on saidimmunosensor, said complex comprising said capture antibody, TSH, andsaid signal antibody; (d) washing uncomplexed signal antibody from thesensor with a wash fluid in no more than one washing step; and (e)detecting a signal associated with said complexed signal antibody.Preferably, the capture antibody and the signal antibody each have adissociation constant (Kd) for TSH of no greater than 1 nM, no greaterthan 0.5 nM, or no greater than 0.15 nM. The sandwich complex preferablyis formed by substantially non-sequential contact of said capture andsignal antibodies with the fluid sample. The sandwich complex preferablyis formed without one or more intervening wash steps directed toameliorating a cross reaction with one or more of FSH, LH and CG. Themethod preferably comprises a single wash step to remove sample andunbound signal antibody from the capture antibody and where the washfluid also comprises a substrate for a reporter molecule. The method mayfurther comprise a step of metering the fluid sample in a meteringchamber to form a metered sample having a volume of from 1 to 500 μL.Optionally, the method comprises a step of bursting a wash fluid pouch,which is in fluid communication with the immunosensor, to release from 5to 500 μL of wash fluid into said conduit. A plurality of air segmentsoptionally may be formed in the wash fluid to facilitate washing of theimmunoassay.

In another embodiment, the invention is to a TSH sandwich immunoassayantibody selection method for non-sequential assays and assays with lowwash fluid cycles, comprising selecting at least two epitope-compatibleantibodies comprising at least one capture antibody and at least onesignal antibody, wherein the capture antibody dissociation constant (Kd)for TSH is less than about 1 nM, less than 0.5 nM or less than 0.15 nM,and wherein the capture antibody Kd value for FSH is greater than 1000nM, greater than 2500 nM or greater than 3000 nM, for LH is greater than500 nM, greater than 1000 nM or greater than 3000 nM, and for CG isgreater than 200 nM, greater than 500 nM or greater than 2500 nM, andwherein the signal antibody Kd for TSH is less than about 1 nM, lessthan 0.5 nM or less than 0.15 nM, and the signal antibody Kd for FSH isgreater than 250 nM or greater than 1000 nM, for LH is greater than 35nM, greater than 200 nM or greater than 250 nM, and for CG is greaterthan 35 nM or greater than 250 nM.

In a preferred aspect, the signal antibody Kd for FSH is preferablygreater than 250 nM or greater than 1000 nM, for LH preferably isgreater than 35 nM, greater than 200 nM or greater than 250 nM, orgreater than 1000 nM, and for CG preferably is greater than 35 nM orgreater than 250 nM. Optionally, the capture antibody Kd value for FSHis preferably greater than 1000 nM, preferably greater than 2500 nM orgreater than 3000 nM, for LH is greater than 500 nM, greater than 1000nM or greater than 3000 nM, and for CG is preferably greater than 200nM, greater than 500 nM or greater than 2500 nM. In terms of ranges, thecapture antibody optionally has a Kd(FSH) of from 1000 to 5000 nM, aKd(LH) of from 500 to 5000 nM, and a Kd(CG) of from 200 to 5000 nM, andthe signal antibody preferably has a Kd(FSH) of from 250 to 5000 nM, aKd(LH) of from 200 to 5000 nM, and a Kd(CG) of from 35 to 5000 nM. Morepreferably the capture antibody has a Kd(LH) of from 1000 to 5000 nM anda Kd(CG) of from 500 to 5000 nM, and more preferably the signal antibodyhas a Kd(LH) of from 35 to 5000 nM. The fluid sample employed preferablycomprises a diluted blood sample, an undiluted blood sample, a dilutedplasma sample, or an undiluted plasma sample.

The capture antibody and the signal antibody, respectively, preferablyare capable of binding to at least two different epitopes on the TSH-βsubunit of TSH. Thus, the capture antibody and the signal antibodypreferably are epitope-compatible antibodies. In a particularlypreferred embodiment, the capture antibody and signal antibody areselected from the following capture/signal antibody pairs 5409/T25C andT25C/5409. The capture antibody, for example, may comprise mousemonoclonal antibody (Biospacific Cat#5409 SPTNE-5) and the signalantibody may comprise mouse monoclonal antibody (FitzgeraldCat#10-T25C).

In one aspect, the capture antibody has a first Kd ratio,Kd(FSH):Kd(TSH), of greater than 2500, e.g., greater than 3000, and thesignal antibody has a second Kd ratio, Kd(FSH):Kd(TSH), of greater than1500, e.g., greater than 1800. Optionally, the first Kd ratio rangesfrom 1500 to 50000, and the second Kd ratio ranges from 1500 to 50000.More preferably, the capture antibody has a first Kd ratio,Kd(FSH):Kd(TSH), of greater than 6000.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood in view of the followingnon-limiting figures.

FIG. 1 is an isometric top view of an immunosensor cartridge cover;

FIG. 2 is an isometric bottom view of an immunosensor cartridge cover;

FIG. 3 is a top view of the layout of a tape gasket for an immunosensorcartridge;

FIG. 4 is an isometric top view of an immunosensor cartridge base;

FIG. 5 is a schematic view of the layout of an immunosensor cartridge;

FIG. 6 is a schematic view of the fluid and air paths within animmunosensor cartridge, including sites for amending fluids with dryreagents;

FIG. 7 shows FRET based binding data for CG and determination of its Kdvalue;

FIG. 8 illustrates magnetic bead capture of FSH and LH;

FIG. 9 is a graph showing reduced cross-reactivity of FSH usingpolystyrene beads;

FIG. 10 is a graph showing reduced cross-reactivity of LH usingpolystyrene beads; and

FIG. 11 is a graph showing reduced cross-reactivity of FSH usingmagnetic beads.

DETAILED DESCRIPTION OF THE INVENTION Introduction

In various embodiments, the present invention relates to immunoassays,devices and methods for performing sandwich Enzyme Linked ImmunosorbentAssays (ELISAs) in the presence of interfering substances, preferablywith limited or no wash step. The invention beneficially permitsaccurate assay results in the presence of these contaminatinginterfering substances. In a first embodiment, the present inventionrelates to devices and methods for conducting thyroid stimulatinghormone (TSH) sandwich immunoassays as well as methods for selectingantibodies for such immunoassays. In a second embodiment, the inventionrelates to immunoassays, in particular to TSH immunoassays, and toassociated devices and methods that employ a single wash step or no washstep. In a third embodiment, the invention relates to immunoassays, inparticular to TSH immunoassays, and associated devices and methods thatemploy scavenger beads coated with antibodies to endogenous contaminantsthat reduce or eliminate interference associated with such contaminants.Alternatively, such antibodies may be covalently bound to the cartridgesurface or may comprise free antibodies in the reaction medium.Optionally, one or more of these embodiments may be combined with oneanother.

TSH Immunoassays and Methods for Selecting Antibodies for TSHImmunoassays

In the first embodiment, the invention relates to immunoassays for TSHand to related devices and methods for conducting such immunoassays. Theinventive immunoassays beneficially provide for the accurate detectionof TSH notwithstanding the presence of the highly related endogenousendocrine glycoprotein hormones, Follicle Stimulating Hormone (FSH),Luteinizing Hormone (LH), and Chorionic gonadotropin (CG). In certaindisease states, these related hormones may be present at significantlyhigh concentrations. The assay is illustrated herein with respect to thei-STAT® immunoassay platform (Abbott Point of Care Inc., Princeton,N.J., USA), which is a low wash volume platform. See jointly owned U.S.Pat. No. 7,419,821, the entirety of which is incorporated herein byreference. In the present specification, the term “low wash” is taken toinclude unitized disposable test devices that incorporate a pouch with arelatively small amount of wash fluid, typically on the order of a fewtens of microliters up to about five milliliters, or similar deviceswhere a similar amount of wash fluid is delivered from an instrumentwith which the device is engaged. In some exemplary embodiments, thedevice and method employ a wash volume of from 5 to 500 μL, e.g., from25 to 250 μL, or from 50 to 150 μL, preferably about 100 μL. It alsoincludes the concept of an assay where the capture antibody, analyte andsignal antibody are mixed together to form the sandwich immunoassay andthen the wash fluid is used to remove sample with unbound analyte andunbound signal antibody from the sandwich that is formed on the captureantibody, which preferably is in some way immobilized, e.g., on a beadthat may be magnetic or on a sensor. These types of devices and systemsare generally associated with testing at the point of patient care.

In another aspect, the invention relates to a method of selectingantibodies for performing a sandwich ELISA assay in a system withlimited wash capabilities. Specifically, antibodies are selected thathave reduced cross-reactivity, which in turn increases analyticalspecificity in a sandwich ELISA. These characteristics are particularlyuseful for low-wash sandwich ELISA assays. As a specific example, theperformance of a TSH assay using an i-STAT® cartridge in the presence ofthe endogenous endocrine glycoprotein hormones, FSH, LH and CG, isdemonstrated herein. By contrast, typical prior commercial systemsassaying TSH utilize a plurality of wash steps to reduce the presence ofthe endogenous endocrine glycoprotein hormones which can be present athigh molar concentrations under certain disease conditions. However, itis apparent that these multiple wash systems would also benefit from theuse of antibodies with these specificity characteristics disclosed inthe present specification, and the present invention should not beconsidered limited to immunoassay systems or devices that employ limitedwash capabilities.

In a preferred embodiment, the two sandwich antibodies are selectedagainst primarily the beta-subunit of the TSH molecule or an epitopeoverlapping with this region exhibiting the desired degree ofspecificity. Ideal characteristics of these two sandwich antibodies arethat they recognize epitopes (preferably different epitopes) of thebeta-subunit of TSH and not exclusively the alpha-subunit of TSH. It iscontemplated that this recognition could involve an epitope spanningboth the alpha and beta subunits that exhibits high affinity to TSH andlow affinity for the other cross-reacting species. Furthermore, theseantibodies preferably exhibit a low Kd for the TSH molecule, whilehaving a high Kd value for the other endogenous glycoprotein hormones,FSH, LH and CG.

As used herein, the term “dissociation constant” or “Kd” refers to theequilibrium constant for the general reaction:

${A + B}\overset{k_{j}}{\underset{k_{- 1}}{\rightleftharpoons}}{AB}$

where A is an antibody and B is an analyte, i.e., antigen. For thisreaction, the dissociation constant or Kd value may be expressed as:

${Kd} = {\frac{\lbrack A\rbrack \times \lbrack B\rbrack}{\lbrack{AB}\rbrack} = \frac{k_{- 1}}{k_{1}}}$

Generally, the lower the dissociation constant, the greater the affinityor degree of binding antibody A has for analyte B. Methods ofdetermining these kinetic values are well known in the art. See, e.g.,Goodrich & Kugel, 2007, Binding and Kinetics for Molecular Biologists,Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y.

In a particularly preferred embodiment, the capture and signalantibodies exhibit a high affinity (low Kd) for TSH. Antibodies thatgenerate poor signal at low concentrations will have high variability(noise) and will be difficult to discriminate from higher measuredcross-reactivity. These antibodies should not be selected as candidateantibodies. In a particularly preferred embodiment, antibodies having aKd for TSH antigen of no greater than 1 nM, e.g., no greater than 0.5nM, no greater than 0.15 nM, or no greater than 0.1 nM, are selected toact as the capture and/or signal antibodies for the immunoassays of thepresent invention.

In another preferred embodiment for the two or more antibodies selectedfor a sandwich hybridization, the antibody having the greatest Kd valuesfor FSH, LH, and CG will be used as the capture antibody. In anotheraspect, if two antibodies are identified as candidates for the captureand signal antibodies, the antibody having the greater Kd value (e.g.,from 5 to 1000% greater, from 20 to 100% greater or from 20 to 50%greater) may be preferred as the capture antibody. In either case, inaddition to having a low Kd value for TSH, e.g., no greater than 1 nM,no greater than 0.5 nM, no greater than 0.15 nM, or no greater than 0.1nM, it is desirable that the antibodies (capture and signal antibodies)have relatively high Kd values for each of the interfering endocrineglycoprotein hormones, FSH, LH and CG.

In a particularly preferred embodiment, the capture antibody has a Kd(FSH) greater than 1000 nM, greater than 2500 nM, greater than 3000 nMor greater than 4000 nM, a Kd (LH) greater than 500 nM, greater than 800nM, greater than 1000 nM, greater than 2500 or greater than 4000 nM, anda Kd (CG) greater than 200 nM, greater than 500 nM, greater than 2500 nMor greater than 4000 nM. In terms of ranges, the capture antibodyoptionally has a Kd (FSH) ranging from 1000 to 5000 nM, from 1500 to4000 nM or from 2500 to 3000 nM, a Kd (LH) ranging from 500 to 5000 nM,from 750 to 1200 nM, from 800 to 1000 nM, from 1000 to 5000 or from 1500to 4000 nM, and a Kd (CG) ranging from 35 to 5000 nM, from 200 to 5000nM, from 210 to 2500 nM or from 220 to 500 nM.

Similarly, the signal antibody preferably has a Kd (FSH) that is greaterthan 250 nM, greater than 1000 nM or greater than 4000 nM, a Kd (LH)that is greater than 35 nM, greater than 200 nM, greater than 250 nM,greater than 1000 nM, greater than 2500 nM or greater than 4000 nM, anda Kd (CG) that is greater than 35 nM, greater than 250 nM or greaterthan 2500 nM. In terms of ranges, the signal antibody optionally has aKd (FSH) ranging from 250 to 5000 nM, from 250 to 2500 nM or from 250 to500 nM, a Kd (LH) ranging from 35 to 5000, from 200 to 5000 nM, from 35to 2500 nM, from 200 to 2500 nM, from 35 to 1000 nM or from 200 to 1000nM, and a Kd (CG) ranging from 35 to 5000 nM, from 35 to 2500 nM or from35 to 1000 nM. In specific embodiments of this method, the TSH bindingspecies preferably are selected from an antibody, a fragment of anantibody (e.g., Fab fragment), a single chain antibody, aptamers,receptors and other specific binding species.

A preferred embodiment of the present invention addresses a TSH sandwichimmunoassay comprising at least two epitope-compatible antibodiescomprising at least one capture antibody and at least one signalantibody, wherein the Kd of both antibodies for TSH is less than (or nogreater than) about 1 nM, e.g., no greater than 0.5 nM, no greater than0.15 nM, or no greater than 0.1 nM, and wherein the capture antibody Kdfor FSH, LH and CG are greater than about 2500, greater than about 500and greater than about 200 nM, respectively, and wherein the signalantibody Kd for FSH, LH and CG are greater than about 250, greater thanabout 200, and greater than about 35 nM, respectively. More preferably,the capture antibody Kd for CG is greater than about 35 nM and thesignal antibody Kd for LH is greater than about 35 nM. In this context,by “epitope compatible” it is meant that the antibodies can selectivelybind to different epitopes on the analyte of interest, as discussedabove. For TSH immunoassays, the antibodies preferably can selectivelybind to different epitopes that are, at least in part, on thebeta-subunit of TSH.

The relative selectivity of the capture and signal antibodies for TSHover FSH, LH and CG may also be characterized in terms of a Kd ratio,i.e., the ratio of Kd(endocrine glycoprotein hormone analogue, e.g.,FSH, LH, CG or an average thereof):Kd(TSH). In this aspect, the largerthe Kd ratio value, the greater the ability of the antibody toselectively bind to TSH over the endocrine glycoprotein hormone analoguein the denominator. In one aspect, for example, the immobilized antibodyhas a first Kd ratio, Kd_(FSH):Kd_(TSH), of greater than 2500, e.g.,greater than 3000 or greater than 5000. Additionally or alternatively,the signal antibody may have a second dissociation constant ratio,Kd_(FSH):Kd_(TSH), of greater than 1500, e.g., greater than 1800 orgreater than 2000. In terms of ranges, the immobilized antibodypreferably has a first Kd ratio, Kd_(FSH):Kd_(TSH), of from 1500 to50000, e.g., from 1800 to 30000 or from 2000 to 28000, and the signalantibody may have a second Kd ratio, Kd_(FSH):Kd_(TSH), of from 1500 to50000, e.g., from 1800 to 30000 or from 1900 to 20000.

As discussed above, the immunoassay preferably employs a pair ofdifferent antibodies that bind to TSH at two non-overlapping epitopes.In a particularly preferred embodiment, the capture antibody comprises aTSH capture mouse monoclonal antibody (e.g., Biospacific Cat#5409SPTNE-5) and the signal antibody comprises a signaling mouse monoclonalantibody (e.g., Fitzgerald Cat#10-T25C). In this aspect, the captureantibody preferably is covalently bound to beads, e.g., latex and/orpolystyrene beads, and the signal antibody is conjugated to a reportermolecule selected from the group consisting of alkaline phosphatase,horseradish peroxidase and a fluorescent or optically absorbing moiety.The detection step is preferably electrochemical, e.g., amperometric,but can also be optical, e.g., fluorescence and absorbance.

The present invention may also be characterized as a ligand bindingassay. For example, in one embodiment, the invention is to a TSHsandwich assay comprising at least one capture ligand and at least onesignal ligand, wherein the dissociation constant of both ligands for TSHis less than (or no greater than) about 1 nM, e.g., no greater than 0.5nM, no greater than 0.15 nM, or no greater than 0.1 nM, and wherein thecapture ligand Kd for FSH, LH and CG are greater than about 2500,greater than about 500 and greater than about 200 nM, respectively, andwherein the signal ligand Kd for FSH, LH and CG are greater than about250, greater than about 200 and greater than about 35 nM, respectively.More preferably, the capture ligand Kd for LH is greater than about 1000nM and for CG is greater than about 500 nM, and the signal ligand Kd forLH is greater than about 35 nM. Here, the ligands may be selected fromthe group consisting of monoclonal antibodies, polyclonal antibodies,fragments of an antibody, aptamers and single chain antibodies.

The invention is also directed to methods for detecting an analyte,e.g., TSH, with an immunoassay that is highly selective for TSH and thatis non-selective for competing endocrine glycoprotein hormone analoguessuch as FSH, LH and CG. For example, in one embodiment, the invention isto a method of performing a whole-blood TSH sandwich assay using atleast two epitope-compatible antibodies comprising at least one captureantibody and at least one signal antibody. The Kd of both antibodies forTSH is less than or about 1 nM, e.g., no greater than 0.5 nM, no greaterthan 0.15 nM, or no greater than 0.1 nM. The capture antibody Kd valuesfor FSH, LH and CG are greater than about 2500, greater than about 500and greater than about 200 nM, respectively, and the signal antibody Kdvalues for FSH, LH and CG are greater than about 250, greater than about200 and greater than about 35 nM, respectively. More preferably, thecapture antibody Kd values for FSH, LH and CG are greater than about1000, greater than about 1000 and greater than about 1000 nMrespectively, and the signal antibody Kd value for LH is greater thanabout 35 nM. The method comprises the steps of: (a) contacting awhole-blood sample with a TSH signal antibody and a TSH capture antibodyimmobilized on an electrochemical sensor to form a sandwich complex, and(b) detecting a signal associated with said complexed signal antibody.

The present invention has utility beyond detection of TSH. For example,in one aspect, the invention is to an analyte sandwich immunoassayperformed in a whole-blood sample comprising at least two antibodies orother ligands comprising at least one capture antibody or ligand and atleast one signal antibody or ligand, wherein the Kd for the analyte withsaid at least two antibodies or ligands is at least a preselected levellower, e.g., 500 times lower, 1000 times lower, or 10,000 times lower,than the Kd for at least two selected known cross-reactants (closelyrelated by protein sequence contaminating antigens). In this aspect, thesandwich preferably is formed by substantially non-sequential antibodyor ligand addition to the sample. The assay may be performed with asingle wash step prior to detection. Here, for example, the targetanalyte can be one of TSH, FSH, LH and CG, and the cross-reactant may beselected from the group consisting of TSH, FSH, LH and CG but is not thetarget analyte. Thus, in other embodiments of the invention, the targetanalyte may be selected from one of FSH, LH and CG rather than TSH.

Low Wash Sandwich Immunoassays

The selective nature of the above-described antibodies or ligands forTSH over competing endocrine glycoprotein hormone analogues such as FSH,LH and CG lends the immunoassay, devices and methods of the inventionparticularly suitable for immunoassays that employ a limited wash step.Thus, the invention is particularly well-suited for implementation inpoint-of-care testing devices, such as the i-STAT® immunoassay platform,which may employ analytical test cartridges or devices that do not havesufficient space for a substantial amount of wash fluid.

With conventional TSH assays, employing multiple and repeated wash stepsis critical for accurate results in order to ameliorate thecross-reactions involving one or more of FSH, LH and CG. According tosome embodiments of the invention, the assay operates where the sandwichis formed by non-sequential antibody addition to a sample, i.e., captureand signal antibodies contact the sample roughly at the same time, andwithout an intervening wash step. The assay optionally includes a singlewash step prior to detection, e.g., a single limited wash step thatremoves blood and unbound signal antibody from the capture antibody.Thus, in a second embodiment, the present invention relates toimmunoassays having a reduced wash step. In one aspect of thisembodiment, a single wash step is used to remove unbound analyte andsignal antibodies from the sandwich assay.

In one aspect of this embodiment, the invention is to a TSH sandwichimmunoassay, comprising at least two epitope-compatible antibodiescomprising at least one capture antibody and at least one signalantibody, wherein the Kd of both antibodies for TSH is less than orabout 1 nM, e.g., no greater than 0.5 nM, no greater than 0.15 nM, or nogreater than 0.1 nM, and wherein the capture antibody Kd for FSH, LH andCG are greater than about 2500 nM, greater than about 500 nM and greaterthan about 200 nM, respectively, and wherein the immunoassay uses asingle wash step. More preferably, the capture antibody Kd for FSH, LHand CG are greater than about 1000 nM, greater than about 1000 nM andgreater than about 1000 nM, respectively. In this context, “a singlewash step” refers to a wash step in which a wash fluid (which preferablyis substantially free of analyte and signal antibodies) is directed tothe immunosensor to remove unbound analyte and unbound signal antibodiesfrom the region of the immunosensor. The amount of wash fluid employedin the single wash step may vary widely, but preferably is less than1000 μL, less than 750 μL, less than 500 μL or less than 250 μL. Thetype of immunosensor employed in the present invention may vary widelyand may be selected, for example, from an electrochemical sensor, asurface acoustic wave sensor, a surface plasmon resonance sensor, athermal sensor, a field effect transistor sensor, an optical sensor, anevanescent wave sensor, a waveguide sensor and the like.

The invention is also directed to methods for detecting an analyte,e.g., TSH, with an immunoassay that employs no more than one washingstep. For example, in one embodiment, the invention is to a method ofperforming a whole-blood TSH sandwich assay using at least twoepitope-compatible antibodies comprising at least one capture antibodyand at least one signal antibody. The Kd of both antibodies for TSH isless than or about 1 nM, e.g., no greater than 0.5 nM, no greater than0.15 nM, or no greater than 0.1 nM. The capture antibody Kd values forFSH, LH and CG are greater than about 2500 nM, greater than about 500 nMand greater than about 200 nM, respectively, and the signal antibody Kdvalues for FSH, LH and CG are greater than about 250 nM, greater thanabout 200 nM and greater than about 35 nM, respectively. Morepreferably, the capture antibody Kd values for FSH, LH and CG aregreater than about 1000 nM, greater than about 1000 nM and greater thanabout 1000 nM, respectively, and the signal antibody Kd value for LH isgreater than about 35 nM. The method comprises the steps of: (a)contacting a whole-blood sample with a TSH signal antibody and a TSHcapture antibody immobilized on an electrochemical sensor to form asandwich complex, (b) washing sample and uncomplexed signal antibodyfrom the sensor, and (c) detecting a signal associated with saidcomplexed signal antibody.

In this embodiment, the washing fluid preferably comprises a fluidcomprising water and one or more additives and should be capable ofwashing the sensors to the desired degree. In one aspect, the washingfluid comprises the enzymatic substrate ANPP (aminonitrophenylphosphate). The washing fluid pH preferably is maintained to be optimalfor the reporter enzyme, which, for alkaline phosphatase, is an optimalalkaline pH of from about 9 to 10. The washing fluid also preferablycomprises a salt to affect electrical conductivity in an electrochemicalassay which is supplied by sodium chloride and magnesium. The magnesiumis also a cofactor of alkaline phosphatase, and may be present at lowconcentrations to enhance enzyme activity. Wash fluids for other enzymebased assays would also contain an appropriate enzymatic substrate,salts and buffers required for enzyme stability and, if electrochemical,for electrical conductivity in a solution, along with any appropriateenzyme cofactors.

Inclusion of Scavenger Beads for Known Interfering Substances

In a third embodiment, the invention is directed to a TSH sandwichimmunoassay that employs one or more scavenger beads and to relateddevices and methods for conducting such immunoassays. To help enhancethe selectivity of the assay for the analyte, e.g. TSH, an embodimentwas devised where scavenger beads comprising for example latex orpolystyrene beads labeled with an antibody to the cross-reactant, e.g.FSH, LH, CG, were added to the mixture that dissolves into the sample.Thus the cross-reactant is presented with comparatively unfavorablebinding sites on the TSH capture antibody and comparatively favorablebinding sites on beads dissolved into the sample.

In one aspect of this embodiment, for example, the invention is to animmunoassay comprising at least two epitope-compatible antibodiescomprising at least one capture antibody and at least one signalantibody. The assay further comprises scavenger beads coated withantibodies (bead antibodies) to FSH, LH and CG, wherein the Kd for eachof said antibodies is less than or about 1 nM, e.g., no greater than 0.5nM, no greater than 0.15 nM, or no greater than 0.1 nM, for each of FSH,LH and CG, and the Kd of each of said bead antibodies for TSH is greaterthan about 250 nM. The Kd for TSH of the capture and signal antibodiesemployed in the immunoassay preferably is less than (or no greater than)about 1 nM, e.g., no greater than 0.5 nM, no greater than 0.15 nM, or nogreater than 0.1 nM, as discussed above.

Thus, in one embodiment, the invention is to a TSH sandwich immunoassaywith at least two epitope-compatible antibodies comprising at least onecapture antibody and at least one signal antibody wherein one or morescavenger beads are used to reduce interference that otherwise may becaused by the competing endocrine glycoprotein hormone analogues FSH, LHand CG. Here, the Kd of the capture and signal antibodies for TSH ispreferably less than or about 1 nM, e.g., no greater than 0.5 nM, nogreater than 0.15 nM, or no greater than 0.1 nM. The added scavengerbeads are coated with bead antibodies to FSH, LH and CG wherein the Kdfor each of these antibodies is preferably less than or about 1 nM,e.g., no greater than 0.5 nM, no greater than 0.15 nM, or no greaterthan 0.1 nM, for each of FSH, LH and CG respectively.

In one aspect, the beads are coated with a single type of antibody thatis selective for each of the competing endocrine glycoprotein hormoneanalogues. In another aspect, the beads are coated with multiple typesof antibodies, wherein a first bead antibody is selective for FSH, asecond bead antibody is selective for LH and a third bead antibody isselective for CG. Other combinations of bead antibodies may be possible,e.g., where a first bead antibody is selective for FSH and LH and asecond bead antibody is selective for CG.

In another aspect, a plurality of different beads may be employed. Forexample, three different types of beads, each having a differentantibody, may be employed to selectively bind to the competing endocrineglycoprotein hormone analogues. For example, the immunoassay, device andmethod may employ first beads, second beads and third beads toselectively bind to FSH, LH and CG, respectively. More specifically, thefirst beads may comprise a first bead antibody selective for FSH, thesecond beads may comprise a second bead antibody selective for LH andthe third beads may comprise a third bead antibody selective for CG.

To avoid TSH preferentially binding to these beads, it is desirable thatthe Kd of each of the bead antibodies (whether on one type of bead or aplurality of types of beads) for TSH is greater than about 250 nM.Preparation of the scavenger beads in terms of antibody labeling followsthe method described herein for the capture antibody for TSH. As withthe embodiments described above, it is preferable that the TSH captureantibody Kd for FSH, LH and CG are greater than about 2500 nM, greaterthan about 500 nM and greater than about 200 nM, respectively. Likewiseit is desirable that the TSH signal antibody Kd for FSH, LH and CG aregreater than about 250 nM, greater than about 200 nM and greater thanabout 35 nM, respectively.

Note that the scavenger beads can be non-magnetic, and remain suspendedin the sample and thus are removed to a waste chamber with the sampleprior to the washing and detection steps. Alternatively, the scavengerbeads may be magnetic and are drawn to a region of the device, by theapplication of a magnetic field, that is away from the TSH capture siteregion, e.g., the TSH immunosensor. Alternatively, the scavenger beadsmay be magnetic, but removed to a waste chamber with or withoutapplication of a magnetic field. Various means for magneticallyretaining magnetic beads in immunosensors are described in U.S. Pat.Appl. No. 61/371,066 to Miller, the entirety of which is incorporatedherein by reference.

The use of magnetic scavenger beads to reduce cross contaminantinterference is illustrated in FIG. 8. As shown, magnetic beads thathave scavenger antibodies selective for LH are added to the sample andselectively bind to LH contained in the sample. A magnetic field, e.g.,from a permanent magnet or from an electromagnet, is applied either inthe device (cartridge) or in the device reader so as to localize themagnetic beads in a region remote from and preferably upstream of thesensor. This advantageously results in reduced interference from thecross-reacting or competing analyte, here LH.

In a preferred embodiment, the sacrificial beads are incorporated into adry reagent coating, which in some embodiments may be the same dryreagent coating that contains the signal antibody. Thus, in oneembodiment, the analysis device includes a dry reagent coating thatcomprises either or both: (a) sacrificial beads suitable forameliorating the effect of one or more of FSH, LH or CG, and/or (b) asignal-generating reagent such as a signal antibody or a labeledanalyte. The dry reagent coating may be formed from a reagent cocktail,which also preferably comprises either or both: (a) sacrificial beadssuitable for ameliorating the effect of one or more of FSH, LH or CG,and/or (b) a signal-generating reagent such as a signal antibody or alabeled analyte. In one aspect, the reagent coating and/or cocktailfurther comprises IgM or fragments thereof for ameliorating interferencecaused by heterophile antibodies, as disclosed in co-pending U.S.application Ser. No. 12/411,325, which is incorporated by reference inits entirety. The surface on which the reagent cocktail is to bedeposited preferably is first Corona treated to provide charged surfacegroups that will promote spreading of the printed cocktail.

In general, the reagent cocktail used to form the dry reagent coatingmay further comprise a water-soluble protein, an amino acid, apolyether, a polymer containing hydroxyl groups, a sugar orcarbohydrate, a salt and optionally a dye molecule. One or more of eachcomponent can be used. In one embodiment, the cocktail contains bovineserum albumin (BSA), glycine, salt, methoxypolyethylene glycol, sucroseand optionally bromophenol blue to provide color that aids visualizingthe printing process. In one embodiment, from 1 to 20 μL of cocktail isprinted onto the desired surface, e.g., within the holding chamber orother conduit, of the analysis device and allowed to air dry (with orwithout heating) before being assembled with its cover. In a preferredaspect, the reagent cocktail and the dry reagent coating formedtherefrom comprise one or more of lactitol, DEAE-dextran, salts such asmagnesium and sodium chloride, IgG/IgM, heparin, surfactant(s) andrhodamine.

The reagent cocktail preferably is formulated as a printable aqueoussolution containing the sacrificial beads and optionally otherinterference-reducing reagents. Upon introduction of a biologicalsample, e.g., blood, the sample preferably mixes with the reagent in afirst step of the assay. The reagent may also include inorganic saltsand surfactants to optimize assay performance with respect to chemicaland fluidic attributes. Other optional additives may include heparin toensure adequate anticoagulation and dyes for visualization of thelocation of the reagent after printing. Also optionally present arestabilizers such as sodium azide for inhibition of microbial growth anda mixture of lactitol and diethylaminoethyl-dextran (Applied EnzymeTechnologies Ltd., Monmouth House, Mamhilad Park, Pontypool, NP4 0HZ UK)for stabilization of proteins. Once deposited in the device, thedeposited reagent may, for example, be dried for 30 to 60 minutes in astream of warm air. In one embodiment, the reagent is printed in thesample inlet of the device using an automated printing instrument anddried to form a sacrificial bead containing reagent coating layer.

In another embodiment, the test cartridge may comprise a plurality ofdry reagent coatings (in which case the coatings may be respectivelyreferred to as a first reagent coating, a second reagent coating, etc.,in order to distinguish them). For example, the sacrificial beads may beincluded in a first reagent coating, which, for example, may be adjacentto a second reagent coating that contains the signal antibody. In thisaspect, the second reagent coating may be located upstream or downstreamof the first reagent coating, although it is preferable for the reagentcoating that contains the signal antibody to be located downstream ofthe reagent coating that contains the sacrificial beads. In a preferredembodiment, the holding chamber is coated with a first reagent coatingthat comprises sacrificial beads and optionally other reagents thatameliorate various forms of interference. In this aspect, a secondreagent coating comprising the signal antibody preferably is locateddownstream of the holding chamber, e.g., immediately upstream of theimmunosensor.

In still other embodiments, the sacrificial beads may not be part of theanalysis device, e.g., cartridge. For example, the sacrificial beads maybe incorporated in a sample collection device, e.g., capillary,Vacutainer™ or syringe. For example, the sacrificial bead coating may beformed on an interior wall of the collection device. Thus, in oneembodiment, the invention is to a kit for performing an immunoassay thatcomprises the sacrificial beads which are first used to amend the bloodsample in a first container or location, and then the sample is passedto a second container or location which has the capture and signalantibodies.

In addition or as an alternative to the beads discussed herein, thesample may be amended with sacrificial beads (optionally opsonized toleukocytes) of the type described in commonly owned U.S. applicationSer. No. 12/620,179 to Campbell et al., the entirety of which isincorporated herein by reference.

Immunosensor Fabrication

As mentioned above, the present invention is best illustrated byreference to the i-STAT® system that uses electrochemical immunosensors.Wafer-level microfabrication of a preferred embodiment of theimmunosensor is as follows. The base electrode consists of a squarearray of 7 μm gold disks on 15 μm centers. The array covers a circularregion approximately 600 μm in diameter, and is achieved byphoto-patterning a thin layer of polyimide of thickness 0.35 μm over asubstrate made from a series of layers comprising Si/SiO₂/TiW/Au. Thearray of 7 μm microelectrodes afford high collection efficiency ofelectroactive species with a reduced contribution from anyelectrochemical background current associated with the capacitance ofthe exposed metal. The inclusion of a PVA layer, e.g., photoformablepolyvinyl alcohol, over the metal significantly enhances the reductionof background currents.

The porous PVA layer preferably is prepared by spin-coating an aqueousmixture of PVA plus a stilbizonium photoactive, cross-linking agent overthe microelectrodes on the wafer. The spin-coating mixture optionallyincludes bovine serum albumin (BSA). The PVA layer may then bephoto-patterned to cover only the region above and around the arrays andpreferably has a thickness of from about 0.2 to 1.0 μm, e.g., from 0.4to 0.8 μm or about 0.6 μm. The immunosensor for different TSH antibodiesmay be made by coating the antibodies onto a bead and applying the beadsto the sensor surface where they adhere.

Using the bead preparation method described below, a droplet of fromabout 30 to 60 nL, e.g., from 35 to 50 nL, or about 40 nL, comprisingfrom about 0.6 to 2.0 wt %, e.g., from about 0.8 to 1.6 wt % or about 1wt % solids in deionized water, may be microdispensed (using, forexample, the method and apparatus of U.S. Pat. No. 5,554,339,incorporated here by reference) onto the photo-patterned porouspolyvinyl alcohol permselective layer covering the sensor and allowed todry. The dried particles should adhere to the porous layer substantiallypreventing their dissolution in the blood sample or the washing fluid.

Capture Bead Fabrication

The capture beads used for the present invention, whether used for theimmobilized capture antibody or the optional scavenger bead antibody,may be formed by a variety of techniques. In a preferred embodiment,carboxylate-modified latex microparticles (Commercially available fromBangs Laboratories Inc. and Seradyn Microparticles Inc.) coated withanti-TSH and anti-HSA are both prepared by the same method. Theparticles preferably are first buffer exchanged by centrifugation,followed by addition of the antibody, which is allowed to passivelyadsorb onto the particles. The carboxyl groups on the particles are thenpreferably activated, e.g., with EDAC in MES buffer at pH 6.2, to formamide bonds to the antibodies. Any bead aggregates may be removed bycentrifugation and the finished beads may be stored frozen, e.g., atabout −80° C.

Cartridge Design

Referring to the figures, an optional cartridge design for use in thepresent invention comprises a cover, FIGS. 1, 2, a base, FIG. 4, and athin-film adhesive gasket, FIG. 3, disposed between the base and thecover. Referring now to FIG. 1, the cover 1 is made of a rigid material,preferably plastic, capable of repetitive deformation at flexible hingeregions 5, 9, 10 without cracking. The cover comprises a lid 2, attachedto the main body of the cover by a flexible hinge 9. In operation, afterintroduction of a sample into the sample holding chamber 34, the lid canbe secured over the entrance to the sample entry port 4, preventingsample leakage, and the lid is held in place by hook 3. The coverfurther comprises two paddles 6, 7, that are moveable relative to thebody of the cover, and which are attached to it by flexible hingeregions 5, 10. In operation, when operated upon by a pump means, paddle6 exerts a force upon an air bladder comprised of cavity 43, which iscovered by thin-film gasket 21, to displace fluids within conduits ofthe cartridge. When operated by a second pump means, paddle 7 exerts aforce upon the gasket 21, which can deform because of slits 22 and 23cut therein. The cartridge is adapted for insertion into a readingapparatus, and therefore has a plurality of mechanical and electricalconnections for this purpose. It should also be apparent that manualoperation of the cartridge is possible. Thus, upon insertion of thecartridge into a reading apparatus, the gasket transmits pressure onto afluid-containing foil pack filled with approximately 130 μL ofanalysis/wash solution (“fluid”) located in cavity 42, rupturing thepackage upon spike 38, and expelling fluid into conduit 39, which isconnected via a short transecting conduit in the base to the sensorconduit. The analysis fluid fills the front of the analysis conduitfirst pushing fluid onto a small opening in the tape gasket that acts asa capillary stop. Other motions of the analyzer mechanism applied to thecartridge may be used to inject one or more air segments into theanalysis fluid at controlled positions within the analysis conduit.These segments are used to help wash the sensor surface and thesurrounding conduit with a minimum of fluid.

The cover further comprises a hole covered by a thin pliable film 8. Inoperation, pressure exerted upon the film expels one or more airsegments into a conduit 20 through a small hole 28 in the gasket.

Referring to FIG. 2, the lower surface of the base further comprisessecond conduit 11, and first conduit 15. Second conduit 11 includes aconstriction 12, which controls fluid flow by providing resistance tothe flow of a fluid. Optional coatings 13, 14 provide hydrophobicsurfaces, which together with gasket holes 31, 32, control fluid flowbetween conduits 11, 15. A recess 17 in the base provides a pathway forair in conduit 34 to pass to conduit 34 through hole 27 in the gasket.

Referring to FIG. 3, thin-film gasket 21 comprises various holes andslits to facilitate transfer of fluid between conduits within the baseand the cover, and to allow the gasket to deform under pressure wherenecessary. Thus, hole 24 permits fluid to flow from conduit 11 intowaste chamber 44; hole 25 comprises a capillary stop between conduits 34and 11; hole 26 permits air to flow between recess 18 and conduit 40;hole 27 provides for air movement between recess 17 and conduit 34; andhole 28 permits fluid to flow from conduit 19 to waste chamber 44 viaoptional closeable valve 41. Holes 30 and 33 permit the immunosensorchip (described above) that are housed within cutaways 35 and 37,respectively, to contact fluid within conduit 15. In a specificembodiment, cutaway 37 houses a ground electrode, and/or acounter-reference electrode, and cutaway 35 houses at least one analytesensor and, optionally, a conductimetric sensor. Hole 29 permits thefilter below access to an external air supply, permitting the generationof air bubbles streamed into the wash fluid flow. The edges of the airbubbles create distinct changes in the wash fluid effecting a partialwashing over the sensors. Typical ELISA assays utilize distinct bufferchanges (rather than partial washing) that permit effective washing ofthe capture sensors.

Referring to FIGS. 3 and 4, conduit 34 is the sample holding chamberthat connects the sample entry port 4 to first conduit 11 in theassembled cartridge via opening 122 in gasket 21. Cutaway 35 houses theanalyte sensor or sensors, or an analyte responsive surface, togetherwith an optional conductimetric sensor or sensors. Cutaway 37 houses aground electrode if needed as a return current path for anelectrochemical sensor, and may also house an optional conductimetricsensor. Cutaway 36 provides a fluid path between gasket holes 31 and 32so that fluid can pass between the first and second conduits. Recess 42houses a fluid-containing package, e.g., a rupturable pouch, in theassembled cartridge that is pierced by spike 38 because of pressureexerted upon paddle 7 upon insertion into a reading apparatus. Fluidfrom the pierced package flows into the second conduit at 39. An airbladder is comprised of recess 43 which is sealed on its upper surfaceby gasket 21. The air bladder is one embodiment of a pump means, and isactuated by pressure applied to paddle 6 which displaces air in conduit40 and thereby displaces the sample from sample chamber 34 into firstconduit 15.

The location at which air enters the sample chamber (gasket hole 27)from the bladder, and the capillary stop 25, together define apredetermined volume of the sample chamber. In some preferredembodiments, the sample is metered to a sample volume of from 1 to 500μL, e.g., from 5 to 200 μL, or from 10 to 50 μL, preferably about 20 μL.An amount of the sample corresponding to this volume is displaced intothe first conduit when paddle 6 is depressed. This arrangement istherefore one possible embodiment of a metering means for delivering ametered amount of an unmetered sample into the conduits of thecartridge.

In the present cartridge, a means for metering a sample segment isprovide in the base plastic part. The segment size is controlled by thesize of the compartment in the base and the position of the capillarystop and air pipe holes in the tape gasket. This volume can be readilyvaried from 1 to 500 μL, e.g., from 1 to 200 μL. Expansion of this rangeof sample sizes is possible within the context of the present invention.

The fluid is pushed through a pre-analytical conduit 11 that can be usedto amend a reagent (e.g., with one or more of scavenger beads, signalantibodies, or soluble molecules) into the sample prior to itspresentation at the sensor conduit 19. Alternatively, one or more of theamending reagents may be located in one or more conduits. For example,the one or more reagents may be located in conduit 34 and/or conduit 15,beyond portion 16. Pushing the sample through the pre-analytical conduitalso serves to introduce tension into the diaphragm pump paddle 7 whichimproves its responsiveness for actuation of fluid displacement.

In some assays, metering is advantageous if quantitation of the analyteis required. A waste chamber 44 is provided for sample and/or fluid thatis expelled from the conduit, to prevent contamination of the outsidesurfaces of the cartridge. A vent 45 connecting the waste chamber to theexternal atmosphere is also provided. A feature of the cartridge is thatonce a sample is loaded, analysis can be completed and the cartridgediscarded without the operator or others contacting the sample.

Referring now to FIG. 5, a schematic diagram of the features of acartridge and components is provided, wherein 51-57 are portions of theconduits and sample chamber that can optionally be coated with dryreagents to amend a sample or fluid. The sample or fluid is passed atleast once over the dry reagent to dissolve it. Reagents used to amendsamples or fluid within the cartridge may include antibody-enzymeconjugates, signal antibodies, blocking agents that prevent eitherspecific or non-specific binding reactions among assay compounds, orsacrificial beads (discussed above). A surface coating that is notsoluble but helps prevent non-specific adsorption of assay components tothe inner surfaces of the cartridges can also be provided.

Within a segment of sample or fluid, an amending substance can bepreferentially dissolved and concentrated within a predetermined regionof the segment. This is achieved through control of the position andmovement of the segment. Thus, for example, if only a portion of asegment, such as the leading edge, is reciprocated over the amendedsubstance, then a high local concentration of the substance can beachieved close to the leading edge. Alternatively, if a homogenousdistribution of the substance is desired, for example, if a knownconcentration of an amending substance is required for a quantitativeanalysis, then further reciprocation of the sample or fluid will resultin mixing and an even distribution.

In specific embodiments, a closeable valve 58 is provided between thefirst conduit and the waste chamber. In one embodiment, valve 58 iscomprised of a dried sponge material that is coated with an impermeablesubstance. In operation, contacting the sponge material with the sampleor a fluid results in swelling of the sponge to fill the cavity 41,thereby substantially blocking further flow of liquid into the wastechamber 44. Furthermore, the wetted valve also blocks the flow of airbetween the first conduit and the waste chamber, which permits the firstpump means connected to the sample chamber to displace fluid within thesecond conduit, and to displace fluid from the second conduit into thefirst conduit in the following manner. After the sample is exposed tothe sensor for a controlled time, the sample is moved into thepost-analytical conduit 19 where it can be amended with another reagent.It can then be moved back to the sensor and a second reaction period canbegin. Alternately, the post-analysis conduit can serve simply toseparate the sample segment from the sensor.

FIG. 6 illustrates the schematic layout of an immunosensor cartridgecomprising three pump means, 61-63. While these pumps have beendescribed in terms of specific embodiments, it will be readilyunderstood that any pump means capable of performing the respectivefunctions of pump means 61-63 may be used within the present invention.Thus, pump means 1, 61, must be capable of displacing the sample fromthe sample holding chamber into the first conduit; pump means 2, 62,must be capable of displacing fluid within the second conduit; and pumpmeans 3, 63, must be capable of inserting at least one segment into thesecond conduit.

Operation

In a preferred embodiment, in a first step, the user draws a bloodsample from the patient (or as in the cases below uses a control fluidwith a known TSH concentration) and inserts a few drops into thecartridge. Once the cartridge is inserted into the instrument, theinstrument controls the rest of the test cycle. The signal antibodypreferably is coated on the wall of a conduit (e.g., holding chamber) inthe cartridge, dissolves into the sample and a portion of the sample ismoved by the pump to the location of the immunosensor. Alternatively,the signal antibody may be disposed in a coating of a conduit in theregion of the immunosensor or elsewhere in the device, but ideally in alocation upstream of the immunosensor. The pump also oscillates thesample to help promote sandwich formation on the sensor. This may takefrom about 1 to 20 minutes, preferably from 2 to 14 minutes, from 4 to12 minutes, or ideally about 8 minutes. Then the pump forces the sampleinto a waste chamber. For embodiments that include a separate wash step,wash fluid from the internal liquid pouch may be delivered via a pumpover the immunosensor to wash away any residual sample and unboundsignal antibody. A portion of the fluid, which also contains the enzymesubstrate, remains in the region of the sensor. Measurement of thecurrent at the immunosensor is then made. Software within the instrumentrecords the values, e.g., currents and other data as shown in the tablesin the examples section, or calculates and displays an actual analyteconcentration.

In operation of the preferred embodiment, which is an amperometricelectrochemical system, the currents associated with oxidation ofp-aminophenol at the immunosensor arising from the activity of ALP, arerecorded by the analyzer. The biochemistry of ALP dephosphorylation ofvarious phosphorylated substrates is well known. The potential at theimmunosensor is poised with respect to a silver-silver chloridereference electrode.

Many types of immunoassay devices and processes have been described andthe following jointly owned patents and applications. A disposablesensing device for successfully measuring analytes in a sample of bloodis disclosed by Lauks in U.S. Pat. No. 5,096,669. It employs a readingapparatus and a cartridge that fits into the reading apparatus for thepurpose of measuring analyte concentrations in a sample of blood. U.S.Pat. No. 7,723,099 to Miller et al. describes an immunoassay device withan immuno-reference electrode; U.S. Pat. No. 7,682,833 to Miller et al.describes an immunoassay device with improved sample closure; U.S. Pat.Appl. Pub. 2004/0018577 to Campbell et al. describes a multiple hybridimmunoassay; U.S. Pat. No. 7,419,821 to Davis et al. describes anapparatus and methods for analyte measurement and immunoassay; and U.S.Pat. Appl. Pub. 2010/0167301 to Collier et al. describes immunoassaycompositions and methods using nucleotide conjugates. Each of thesepatents and applications is incorporated herein by reference in itsentirety.

It is well known in the art that immunoassays are susceptible to variousforms of interferences. Jointly-owned pending U.S. application Ser. No.12/411,325 (the “'325 application”), for example, addresses amelioratinginterferences from heterophile antibodies by the inclusion of IgM intoan IgG reagent cocktail. The '325 application is incorporated herein byreference in its entirety.

Empirical Versus Theoretical Approaches to Antibody Selection for LowWash TSH Assays

For a TSH assay, the related hormones can potentially be found at highmolar concentrations compared to TSH in a number of clinical situations.Therefore, FSH, LH and CG are typically added to a TSH assay at thelevels of 500, 500, and 200,000 mIU/mL, respectively, to simulate a realsample in the presence of high concentrations of cross-reacting species(Architect Product Data Insert, Abbott, Chicago, Ill.). Theseconcentrations for TSH, FSH, LH and CG are approximately 0.96, 3000, 760and 436,000 pM, respectively.

Theoretical Cross Reactivity

Once the equilibrium dissociation constants have been measured,theoretical calculations can be performed in order to compareanticipated cross reactivity based on the obtained dissociationconstants. These calculations cannot exactly predict the outcome of theactual assay, but may be used as a guide to ensure that the affinity ofthe antibodies will suffice for performing the assay, and that selectedantibodies will perform significantly better when tested in low washcartridge experiments. These calculations can also aid development ofthe assay by identifying unexpected assay results during the prototypephase of development. See, e.g., Table 1, below.

Calculations were performed by assuming a two-step assay and prior to awash procedure, the two-step assay comprising: (1) a capture step and(2) a detection step. In the capture step it is assumed that all fourhormones bind with the capture antibody and reach equilibrium. Theconcentration of each hormone bound to the capture antibody after thisstep is considered the captured hormone. In the detection step, thecaptured hormone is assumed to reach equilibrium in binding to thedetection antibody. The concentration of each hormone bound to thedetection antibody after this second step is the detected concentration.The cross reactivity is then calculated by dividing the detectedconcentration of the interfering hormone by the detected concentrationof TSH and multiplying by 100%. The measured dissociation constants forthe antibodies given in Table 8 were used for the calculations. Theconcentrations for TSH, FSH, LH and CG are approximately 1, 3000, 760and 436,000 pM, respectively. These values are comparable to thosetypically added to a TSH assay in order to challenge the assay. Thiscalculation is used to confirm that the antibodies selected are animprovement over the control antibodies. The calculation assumes 100 pMcapture antibody and 100 pM detection antibody.

Since the model cannot exactly predict the outcome in an actual assay asother factors such as determining a compatible antibody pair or theimpact of mass transport for the capture antibody, the data is simplyused as a guide to confirm that the antibodies have merit, and estimatetarget dissociation constants. Therefore, in order to compare theantibodies, the values determined from the cross reactivity calculationare compared in the following manner. The control cross reactivityvalues are defined by performing the above calculation using antibodiesTSH Ab 544 and 414B. The best value for cross reactivity obtained forthese control antibodies is taken to be the cross-reactivity which mustbe improved upon. This value is indicated with an asterisk for eachhormone in Table 1, below. Any candidate antibody pair should have lowercross-reactivity than these values in both configurations. From the datain the table, we see that the combination of 414B and 5409 has acondition in which the cross reactivity to FSH and CG are above thecontrol values. This result suggests that this combination may not bethe best pair for a TSH assay. The table also indicates thatcombinations of 5409, ME130 and T25C yield values of cross reactivitybelow the control values in either combination, for all hormones. Thisindicates that these antibodies may be good candidates for use in theassay and were investigated further.

TABLE 1 CROSS-REACTIVITY PERCENT-CALCULATED Capture Ab Signal Ab FSH LHCG 5409 ME130    0.04 0.01    40 ME130 5409    0.04 0.01      7.3 5409T25C    0.02 0.01    75 T25C 5409    0.02 0.01    12 414B 5409    0.730.03    6 5409 414B  11 0.03   7800 Control TSH Ab 544 414B 145  0.74*85,200 414B TSH Ab 544  10* 0.79    120* *Denotes poor sandwich antibodypairs.

The present invention will be better understood in view of the followingnon-limiting examples.

Example 1 Initial Antibody Screening

Sixty-seven antibody preparations were obtained (Table 2) and testedwith a nitrocellulose dot blot assay. Additionally, TSH Antibody 544 andSeradyn anti-alpha-LH monoclonal antibody (Seradyn, Cat#MIT0414B) wereused in these studies as benchmark cross-reacting pair of antibodies. Toprovide assay standards, the antigens TSH (Cat#T9265, Sigma, St. Louis,Mo.), FSH (Cat#F4021, Sigma, St. Louis, Mo.), LH (Cat#L5259, Sigma, St.Louis, Mo.) and CG (Cat#C0434, Sigma, St. Louis, Mo.) were resuspendedin ⅕ PBS buffer to an approximate concentration of 35,000 uIU/mL,700,000 mIU/mL, 250,000 mIU/mL, 2×10⁷ mIU/mL for TSH, FSH, LH, and CG,respectively. One μL of each antigen was spotted and dried onto a smallstrip of nitrocellulose. The nitrocellulose strip was first blocked with1% powdered milk in PBS, then individual strips were incubated with eachof the different antibody preparations. The strips were then washed withPBS, 0.05% Tween-20, followed by addition of Goat anti-mouse IgG H+L ALPsecondary conjugate (Cat#4751-1806, Kirkegaard & Perry, Gaithersburg,Md., USA) and where appropriate anti-rabbit, sheep, and goat IgG H+L ALPconjugates were used for certain polyclonals, then the BCIP/NBT alkalinephosphatase substrate (1-component) (Cat#50-81-07, Kirkegaard & Perry,Gaithersburg, Md., USA) was added. The reaction was stopped by rinsingin deionized water.

TABLE 2 LIST OF SCREENED ANTIBODIES Vendor Catalogue # ImmunoreagentsMuxHu-012A-Q MuxHu-012B-Q MuxHu-012C-C GtxHu-012-D Maine BiotechnologyMAB128P Services MAB129P MAB130P MAB131P MAB132P Biospacific 5405 5409G-109-C S-109-C 5401 5403 5404 Hytest Anti-h TSH 11E4 Anti-h TSH 7G12Anti-h TSH 10C7 Anti-h TSH 5E8 Anti-h TSH TSB1 Anti-h TSH TSB4 SantaCruz Biotech sc-7813 sc-7814 sc-7815  sc-28917 Seradyn M1T0401 M1T0406M1T0409 M1T0412 M1T0414 Meridian Life Sciences MAT04-004 clone 057-11004MAT04-410 clone 204-12410 MAT04-176 clone 090-11176 MAT04-127 clone090-10127 MDT04-005 clone 057-11005 MAT04-005 clone 057-11005 MAT04-252clone 204-12252 MAT04-006 clone 057-11006 MAT04-003 clone 057-11003MCT04-001 clone 057-11001 ThermoScientific Pierce MA1-82908 MA1-82909MA1-83492 Immunoreagents GtxHu-012-D ShxHu-012-D Biospacific G-109-CS-109-C Abd Serotec 8920-0456 8920-0600 8920-0609 0200-0064 clone 1540200-0065 clone 155 8926-0511 Meridian Life Sciences D92409G ChromaprobeUHS115 AbCam ab9390 ME-130 ab1989 ME-128 ab9239 ME-131 Fitzgerald10C-CR2151M3 10C-CR2151M4 10C-CR2151M5 10C-CR2151M6 10-T25D 10-T25A10R-T128A 10-T15A 10-T15B 10-T15C 10-T25B 10-T25C

Antibodies selected for further evaluation were determined byidentifying those strips exhibiting good signal with the TSH antigen,and low signal with FSH, LH, and CG. This resulted in twenty-fivecandidate antibodies for further evaluation (Table 3), including TSH Ab544 and Seradyn MIT0414B as controls.

TABLE 3 CANDIDATE ANTIBODIES FOR FURTHER CROSS-REACTIVITY ANALYSISVendor ID Abbott Diagnostics Division TSH Ab 544 Seradyn (Thermo-Fisher)MIT0414B Hytest 5E8 Hytest 7G12 Abd Serotec 8920-0600 Abd Serotec0200-0064 Meridian Life Sciences MAT04-127 Meridian Life SciencesMAT04-252 Abcam ME-128 Thermoscientific Pierce MA1-82908 Abcam ME-130Abcam ME-131 Biospacific 5404 Biospacific 5409 Fitzgerald 10-T25BFitzgerald 10-T25C Fitzgerald 10C-CR2151M3 Fitzgerald 10C-CR2151M4Fitzgerald 10-T25D Fitzgerald 10-T25A Fitzgerald 10R-T128A Fitzgerald10-T15A Fitzgerald 10-T15C Fitzgerald 10C-CR2151M5 Fitzgerald 10-T15B

The twenty-five candidate antibodies were then used to generate bothcapture beads and ALP conjugates as described in Examples 10 and 11,below. These reagents were built into i-STAT® cartridges and thenscreened for cross-reactivity using the test cycle described herein. Theconcentrations of the antigens (control test fluids) were approximately0.4 mIU/L, 500 mIU/L, 500 mIU/L, and 200,000 mIU/L for TSH(ThermoFisher, Cat#ABT0315), FSH, LH and CG, respectively. FSH, LH andCG reagents were from the same source as described above. The assayswere performed with TSH alone, as well as with TSH in combination witheach CG, FSH, and LH individually. The percent cross reactivity wascalculated by the difference between the electrochemical signalgenerated from TSH with one of the cross reacting antigens, less the TSHsignal by itself divided by the signal generated by TSH alone.

This screening led to four candidate antibodies used for additionalevaluation (Table 4). The TSH Ab 544 and MIT0414B antibodies were usedfor comparison studies, as high cross reacting controls. Vendor supplieddata from the antibodies with the lowest relative cross reactivity arelisted in Table 4. It should be noted that these had the lowest levelsof cross reactivity with the antigen samples used. It was lateridentified and confirmed by testing these interfering antigens using theAbbott Architect i2000SR instrument (Chicago, Ill., USA) that theseantigens contained trace amounts of TSH antigen, leading to an observedhigher level of cross reactivity which was not seen in later experimentsas higher purity antigens were used (purchased from Fitzgerald, NorthActon, Mass.). As the cross reactivity levels were lower relative to theother antibodies tested, these antibodies were anticipated to have thebest cross reacting performance and were further analyzed by othertechniques described below.

TABLE 4 VENDOR INFORMATION FOR SELECTED ANTIBODIES Ka Company Cat#Species Type Clone Immunogen Epitope (L/mol) AbCAM ME-130 Mus mAb Ab9390Full length Beta-TSH 2 × 10¹⁰ (ME130) IgG1 human TSH Fitzgerald 10C- MusmAb 157155 Human TSH 2 × 10¹⁰ CR2151M4 IgG1 Pituitary TSH (M4)Fitzgerald 10-T25C Mus mAb M94206 Human Beta-TSH 2 × 10¹⁰ (T25C) IgG1Pituitary TSH Biospacific 5409 Mus mAb N/A N/A TSH 9.3 × 10⁸   SPTNE-5IgG1 (5409)

Example 2 FRET Epitope Mapping

Antibody Pair Compatibility was determined by Forster Resonance EnergyTransfer (FRET) based competition assays. Approximately 500 to 1000 μgof each endocrine glycoprotein hormone (TSH (Cat#T9265, Sigma, St.Louis, Mo.), FSH (Cat#30R-AF020, Fitzgerald, North Acton, Mass.), LH(Cat#30-AL15, Fitzgerald, North Acton, Mass.), and CG (Cat#30R-AC048,Fitzgerald, North Acton, Mass.) were labeled with Alexa-Fluor® 488Carboxylic Acid succinimidyl ester (Cat#A20100, Invitrogen, Carlsbad,Calif.). The antibodies were labeled with BHQ-10 Carboxylic Acidsuccinimidyl ester (Cat#BHQ-10S, Biosearch Technologies, Inc., Novato,Calif.) as a fluorescent quenching moiety. The proteins were labeledaccording to Ruan et al. (2009, Analytical Biochemistry, vol 393:196).

The antibody pairs were selected based on their quenching capability inthe presence of an unlabeled antibody. Excess amount of the sixantibodies was first incubated with the fluorescently labeled antigen,and then each sample was divided into six test tubes. In the six testtubes, one of the six BHQ labeled antibodies was added. Thus, a 6×6reaction matrix was generated as shown in Table 5. The fluorescenceintensity from each test tube was measured before and after the additionof the BHQ-labeled antibody. Significant change in fluorescenceintensity indicated positive sandwich pairing. No change or littlechange in fluorescence intensity indicated the presence of the unlabeledantibody blocking the binding of the BHQ labeled antibody to the sameanalyte. Fluorescence was normalized to a value of 1.0 for same pairs.Table 5 provides relative fluorescence values.

TABLE 5 BHQ labeled antibodies TSH Ab 5409 T25C ME130 M4 544 414BUnlabelled 5409 1*   0.78 0.78 0.90* 0.93* 0.88* Antibodies T25C 0.95*1*    0.97* 0.92* 0.97* 0.97* ME130 0.82   0.88* 1*   0.77  0.82* 0.99*M4 0.94* 0.74 0.76 1*   0.98* 0.90* TSH Ab 0.96* 0.71 0.72 0.92* 1*  0.89* 544 414B 0.81  0.76  0.86* 0.75  0.83* 1*   *Denotes poor sandwichantibody pairs.

Example 3 Epitope Mapping with i-STAT® Cartridge

Both polystyrene beads and ALP conjugates were generated for each of thesix antibodies (described in Example 2). The cartridges were tested with0.4 mIU/L TSH (recombinant TSH, ThermoFisher, Cat#ABT0315, Fremont,Calif., USA) generating an electrochemical current (nA). Table 6 lists anumber of antibody pairs which indicate compatible antibody pairs. Sameantibody pair combinations were not tested. For example, 414B recognizesthe alpha subunit which would be anticipated to have high crossreactivity due to the biology of the binding and was not tested in thecartridge. It should be appreciated that there is a potential forepitope blocking by labeling the antibody with either fluorescent tagsor fluorescent quenching moieties, which could account for thedifference between both the FRET and sandwich ELISA methods,particularly for T25C with BHQ labeled 5409 and M4. Antibody pairs weretested in the electrochemical immunoassay system of the i-STAT cartridgeusing TSH antigen at 30 mIU/L concentration. Values in Table 6 are in nAof current measured.

TABLE 6 Detection Conjugate Antibody TSH Ab 5409 T25C ME130 M4 544 414BCap- 5409 NT* 34.59 53.64  15.19* 1.41* NT* ture T25C 49.71 NT*  0.68*40.90 0.83* 0.82* Anti- ME130 54.66  −0.10* NT* 50.53 10.76*  NT* bodyM4  20.67* 22.52 39.92 NT* 0.00* NT* TSH Ab  7.98* 22.43 34.85  0.11*NT* 25.0   544 414B 40.29 NT* NT* 41.27 10.15*  NT* *Denotes poorantibody pairs. NT = Not tested

Antibody pairs for cross reactivity evaluation are listed in Table 7based on the FRET competition assay and i-STAT® cartridge data.

TABLE 7 POSSIBLE ANTIBODY PAIRS FOR FURTHER EVALUATION Capture Signal5409 T25C 5409 ME130 T25C 5409 T25C M4 ME130 5409 ME130 M4 M4 T25C M4ME130 TSH Ab 544 T25C TSH Ab 544 ME130 TSH Ab 544 414B 414B 5409 414B M4

Example 4 Kd Calculations

Approximately 500 to 1000 μg of each endocrine glycoprotein hormone (TSH(Cat#T9265, Sigma, St. Louis, Mo.), FSH (Cat#30R-AF020, Fitzgerald,North Acton, Mass.), LH (Cat#30-AL15, Fitzgerald, North Acton, Mass.),and CG (Cat#30R-AC048, Fitzgerald, North Acton, Mass.) were labeled withAlexa-Fluor® 488 Carboxylic Acid succinimidyl ester (Cat#A20100,Invitrogen, Carlsbad, Calif.). The antigen was also labeled with BHQ-10Carboxylic Acid succinimidyl ester (Cat#BHQ-10S, Biosearch Technologies,Inc., Novato, Calif.) as a fluorescent quenching moiety. The proteinswere labeled according to Ruan et al. (2009, Analytical Biochemistry,vol 393:196).

The addition of approximately 100 to 250 nM concentration of BHQconjugated antibody with from 100 pmol for TSH to about 1000 pmol forLH, CG and FSH of each of the Alexa-Fluor® 488 labeled antigens wasperformed using an SLM-Aminco Model SLM 8100 spectrofluorometeraccording to Ruan et al, (2009, Analytical Biochemistry, vol 393:196).The data was analyzed with IDL software (ITT, Boulder, Colo.) togenerate a Kd value (nM). An example of the data is seen in FIG. 7. Asummary of the Kd data is found in Table 8. ME130, M4 and 414B did nothave the antibody characteristics of Tables 9 and 11 and therefore donot exhibit the low level of cross reactivity. Table 8 provides Kdbinding data for six antibodies using 4 different antigens; all valuesare in nM.

TABLE 8 Antibody TSH FSH LH CG 5409 0.1 1000 1000 500 T25C 0.08 300 3540 ME130 0.04 70 54 45 M4 0.18 210 300 554 TSH Ab 544 1.31 1000 1000 210414B 0.04 0.1 0.05 0.05

It is important that the antibody generate sufficient signal as thecross reactivity levels are higher for low signal. Therefore, antibodieswithout sufficiently low Kd values (high affinity for TSH) in Table 9were not considered for cross-reactivity. This generated the new list ofpossible antibody pairs for cross reactivity testing (Table 10).

TABLE 9 PREFERRED ANTIBODY TSH BINDING CHARACTERISTICS TO GENERATE LOWCROSS REACTIVITY Kd TSH (nM) Capture/Signal ≦0.15 Antibody

TABLE 10 REMAINING POSSIBLE ANTIBODY PAIRS WITH ADEQUATE TSH ANTIGENAFFINITY Capture Signal 5409 T25C 5409 ME130 T25C 5409 ME130 5409 414B5409

Those antibodies having the antibody Kd characteristics found in Table11 were anticipated to have low cross-reactivity.

TABLE 11 PREFERRED ANTIBODY CHARACTERISTICS TO GENERATE LOW CROSSREACTIVITY Kd FSH (nM) Kd LH (nM) Kd CG (nM) Capture >1000 >1000 >500Antibody Signal Antibody >250 >35 >35

Example 5 i-STAT® Cross-Reactivity Data

Conjugated antibody bead preparations and Alkaline Phosphatase (ALP)conjugate combinations were built into i-STAT® immunoassay cartridges.

Based on compatible antibody pairs (Table 7), appropriate Kd values forTSH (Table 9), and high Kd values for FSH, LH and CG (Table 11), thecombinations of Table 10 were tested for cross reactivity as describedin Example 1, and are summarized in Table 12. Antibody pairs exhibitingcross reactivity greater than 5% for any of the interfering antigenswere indicated with an asterisk (*). Only 5409 (capture antibody) andT25C (signal antibody) exhibited cross reactivity of less than 5% asthey are the only antibodies with the characteristics described inTables 9 and 11.

The antibody combination of 5409 used as a capture with T25C used as adetection antibody were the only pair capable of conferring lowcross-reactivity due to their antigen binding characteristics, alongwith their ability to recognize compatible epitopes. ME130 had betterbinding affinity to TSH, which was exhibited with higher amperometriccurrent generation. Antibodies exhibiting higher amperometric signalsgenerated lower cross reactivity compared to those antibody pairsgenerating lower amperometric signals, which were more problematic todifferentiate from the noise in the system. Table 12 indicates antigencross reactivity in the i-STAT® cartridge format using 0.4 mIU/L TSH andadding all of the antigen concentrations. Only compatible antibody pairsdetermined previously were selected. M4 was removed from the selectionas it had poor affinity to TSH, and generated low signal in thecartridge assay compared to other antibody combinations (Table 6 andTable 8).

TABLE 12 Capture Ab Detection Ab FSH CR (%) LH CR (%) CG CR (%) 5409ME130*  2.43*  10.08*  0.78* 5409 T25C  0.65  3.76  0.96 T25C 5409* 6.25*  9.89*  13.72* ME130 5409*  3.90*  11.05*  11.84* 414B 5409*260.5*  347.64*  84.6* Control 414B TSH Ab 544* 219.21* 467.21*  39.17*94544 414B*  7.15*  11.75* 117.14* *Denotes pairs with less desirablecross-reactivity above 5%.

Example 6 Anti-FSH 95784 Bead Preparation

The Anti-FSH 95784 beads were prepared as follows: 15 mg of 1.01 μmcarboxylated polystyrene microparticles (10% weight/volume) (part#PCO4N, Bangs Laboratories Inc., USA), were reacted with 1.2 mg ofAnti-FSH 95784 in 25 mM 2-(N-morpholino)ethanesulfonic acid (MES buffer,pH 6.2) for 15 minutes, and then were centrifuged to remove thesupernatant. After resuspension of the pellet in 25 mM MES buffer, 10 mMcarbodiimide (EDAC) was added to the sample and reacted for 2 hours at4° C. This was followed by centrifuging the sample, washing the pelletwith ⅕ physiological phosphate buffer twice. A formulated sample with10% solids in phosphate buffer including 0.05% Tween 20 was stored forfurther use.

These beads were used in the assembly of FSH detecting cartridges wherethe TSH capture antibody is replaced with FSH conjugated beads. Thebeads were formulated to 3.2% solids in ⅕ physiological phosphatebuffer, including 25% protein stabilization solution (Cat#Q2030529P1,Gwent Group, Pontypool, United Kingdom). A formulation of 0.8% solid,0.8% protein stabilization solution in 0.08% Tween-20 was printed onchips and built into FSH cartridges.

Approximately 5 to 10 mg/mL (or 0.5 to 1% solid) of FSH beads werespiked into the test sample containing approximately 500 mIU/mL FSH,mixed vertically with pipette and immediately injected into the sampleinlet of the FSH detecting cartridge. The beads can also be printed ontothe sample inlet of the cartridge and dissolved into the sample later asthe sample is introduced into the cartridge.

Example 7 LH Bead Preparation

LH polystyrene beads were prepared similar to Example 6, above, exceptthat Biospacific LH 5304 antibody was used in place of Anti-FSH 95784.And as in Example 6, above, these beads were used to make LH detectingcartridges.

Example 8 FSH Magnetic Bead Preparation

FSH magnetic beads were prepared as follows: 15 mg of 0.70 μm superparamagnetic microsphere (10% weight/volume) (Catalog # MC04, BangsLaboratories Inc, USA) were reacted with 30 mM carbodiimide (EDAC) in 50mM 2-(N-morpholino)ethanesulfonic acid (MES buffer, pH 6.2) in acentrifuge tube for 15 minutes. The tube was placed on a magnet toattract the magnetic beads to the side of the magnet while aspiratingout the supernatant. After two washes with 50 mM MES, 1.2 mg of Anti-FSH95784 (Abbott Diagnostics Division) was reacted with the beads for 90minutes at 4° C. The beads were separated from the supernatant byplacing a magnet to the side of the centrifuge tube, and then remove thesupernatant. This was followed by washing the pellets with ⅕physiological phosphate buffer twice. A formulated sample with 5% solidsin phosphate buffer including 0.05% Tween 20 was stored for further use.

Approximately 5 to 10 mg/mL (or 0.5 to 1% solid) of FSH bead was spikedinto the test sample containing approximately 500 mIU/mL FSH, mixedvertically with pipette and immediately injected into the cartridgesample inlet. The beads can also be printed onto the sample inlet of thecartridge and dissolved into the sample later as the sample isintroduced into the cartridge.

Example 9 LH Magnetic Bead Preparation

LH magnetic beads were prepared similar to Example 8 above except thatBiospacific LH 5304 monoclonal antibody was used in place of Anti-FSH95784.

Example 10 TSH Bead Preparation

TSH 5409ANA beads were prepared as follows: 15 mg of 0.2 μm carboxylatedmicroparticles (10% weight/volume) (part#13000550100390, Seradyn,Indianapolis, Ind., USA), were reacted with 1.2 mg of TSH 5409 mAb in 25mM 2-(N-morpholino)ethanesulfonic acid (MES buffer, pH 6.2) for 15minutes, and then were centrifuged to remove the supernatant. Afterresuspension of the pellet in 25 mM MES buffer, 10 mM carbodiimide(EDAC) was added to the sample and reacted for 2 hours at 4° C. This wasfollowed by centrifuging the sample, washing the pellet with ⅕physiological phosphate buffer twice. A formulated sample with 3.2%solids in ⅕ physiological phosphate buffer, including a proteinstabilization solution (Cat#Q2030529P1, Gwent Group, Pontypool, UnitedKingdom) was stored for further use. These beads were printed on chipsused to build TSH cartridges.

TSH reference bead preparation was as follows: The process was the sameas 5409ANA bead process except using anti-HSA antibody (HyTest Ltd, Cat4T24 Mab 1C8, Joukahaisenkatu, Turku, Finland) in the reaction insteadof 5409 mAb. The use of reference beads in immunosensor manufacture andoperation is described in U.S. Pat. No. 7,732,099, the entirety of whichis incorporated herein by reference. Here, an immuno-reference sensor isused to subtract a signal arising from non-specific binding of thesignal antibody to the immunosensor.

Example 11A TSH Signal Antibody Conjugate Synthesis

A preferred embodiment of the signal antibody conjugate synthesis is asfollows: TSH T25C conjugate preparation used pepsin digested T25C wholeantibody to make T25C F(ab)2′ in 0.1 M Citrate Buffer (pH 3.5) at 37° C.Purification of the T25C F(ab)2′ fraction was done by using a S-300 sizeexclusion column (GE Healthcare, SE-751 84 Uppsala, Sweden).Monoethanolamine hydrochloride (MEA) was used to reduce T25C F(ab)2′ toFab-SH, which was then reacted with LC-SMCC(Succinimidyl-4-[N-Maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate])and activated single molecule ALP (alkaline phosphatase) at 4° C.overnight. A size exclusion column was then used to purify the conjugatefraction and formulate it into a ⅕ physiological phosphate-bufferedprotein stabilization solution. This was then stored frozen at −80° C.for further use.

Example 11B LH and FSH Signal Antibody Conjugate Synthesis

For the FSH and LH experiments, the 414B antibody recognizing thealpha-subunit was replaced with T25C described in Example 11A, above.This antibody was used for the scavenger bead experiments as thedetection antibody.

Example 12 Scavenger or Sacrificial Beads

The utility of the scavenger bead approach is shown in FIGS. 9-11. Asshown in FIG. 9, the use of polystyrene beads significantly reduced FSHinterference from more than 100% cross reactivity to less than 15%. FIG.9 is an experiment wherein FSH beads (Example 6) are built into FSHdetecting cartridges. The sample contains extremely high concentrations(500 mIU/mL) of FSH. The signal with no addition of FSH scavenger beadswas assigned a value of 100%. After the addition of FSH scavenger beads,the signal was reduced to 15%.

FIG. 10 focuses on the effect of LH scavenger beads from example 7. Forthe antibody combination LH capture and 414B described in Examples 7 and11B, the LH cross-reactivity is 100% without the scavenger beads,whereas adding the scavenger beads to the assay cycle reduces theinterference to 4%.

In the foregoing example, the LH antibody was the monoclonal antibodymus beta-LH, Catalog #5304 SP-5. Approximately 0.5 mg/mL of beads wereused. The FSH mAb was Anti-FSH 95784 SP-5. Table 13, below, providesadditional information concerning various commercially availablematerials that may be employed as scavenger beads according to thisembodiment of the invention.

TABLE 13 Commercially Available Antibodies Company Cat# Species TypeClone Immunogen Epitope Use As Biospacific Anti-FSH Mus mAb N/A N/ABeta- Scavenger 95784 SP-5 IgG1 FSH Biospacific 5304 SP-5 Mus mAb N/AN/A Beta-LH Scavenger IgG1 Human Seradyn MIT0414B Mus mAb BB3.1.3Luteinizing Alpha Signal IgG1 Hormone Subunits Abbott 95784 N/A N/A N/AN/A Beta- Scavenger Diagnostics FSH Division

FIG. 11 illustrates a significant reduction in FSH cross reactivity offrom about 100% to less than 10% by employing magnetic beads. In thisexperiment, FSH detecting cartridges were built containing FSHpolystyrene capture beads (Example 6), a Nickel coated (Nd₂Fe₄B)permanent magnet was built into the cartridge as shown in FIG. 8. Also,super paramagnetic FSH scavenger beads (Example 8) were printed on thesample inlet port (FIG. 5 sample inlet). With these componentmodifications, the cartridge was built similar to the standardprocedures. The experiment was executed by adding approximately 20 μL ofAbbott Architect 0 TSH Calibrator (Chicago, Ill.) sample spiked with 500mIU/mL of FSH.

The analyzer executed the steps in the assay with the cartridge and theresulting current generated in the assay was converted to 100% for theno scavenger bead cartridge test, and the sample with scavenger beadsprinted in the cartridge was calculated based on the no scavenger beadresult, showing a significant reduction in signal, confirming thecapability of the scavenger beads to reduce background in the assay.

Example 13 Screening for Substitute Monoclonal Antibody

As an alternative embodiment, eleven antibody preparations were obtainedfrom a stock of TSH antibody cell lines at Abbott Diagnostic Division(ADD) as potential substitutes for the T25C and/or 5409 antibodies. Thescreening approach for a potential substitute monoclonal antibody toreplace the T25C and/or 5409 antibodies was based on the hypothesis thatif the same epitope was found in the uncharacterized antibody, that theyshould potentially possess similar properties to the T25C and/or 5409antibodies.

TABLE 14 CANDIDATE ANTIBODIES FOR SUBSTITUTION Vendor ID Ab EpitopeAbbott Diagnostics Division Clone 10-542-594 N/A Abbott DiagnosticsDivision Clone 10-1064-137 5409 Abbott Diagnostics Division Clone10-1332-190 N/A Abbott Diagnostics Division Clone 10-541-173 N/A AbbottDiagnostics Division Clone 10-518-308 5409 Abbott Diagnostics DivisionClone 10-880-320 5409 Abbott Diagnostics Division Clone 10-755-148 5409Abbott Diagnostics Division Clone 10-1179-456 T25C Abbott DiagnosticsDivision Clone 10-266-130 5409 Abbott Diagnostics Division Clone10-529-114 5409 Abbott Diagnostics Division Clone 10-542-219 N/A

Approximately 0.1 ug ADD TSH whole mAb candidates were spiken along with39 mIU/L TSH sample, mixed well and tested on the i-STAT® immunoassaycartridges with 5409 as the capture antibody and the T25C as the labelantibody. Two antibody clones, 10-518-308 and 10-1179-45 (Table 14)demonstrated significant decrease of the detection signal, whichindicated that they were competing with either the 5409 or the T25Cantibody.

Once the antibody clones 10-518-308 and 10-1179-45 were identified aspotential replacements for 5409 and/or T25C, the next testing step wasto make beads and conjugate out of the monoclonal antibodies and todetermine if they would form a usable sandwich (compatible antibodyepitope pair) and determine whether they exhibited a good signal withTSH, and good selectivity against FSH, LH and CG.

The ADD TSH antibody 10-518-308 was made into ANA beads, printed on asensor and built into no conjugate cartridges. The ADD TSH 10-1179-45was digested into F(ab)2′, reduced to Fab fragment and furtherconjugated with ALP to form the conjugate for the cartridge test. A 5409ANA and T25C conjugate were used as control for performance comparison.There are four cartridge combinations for the following step in theevaluation.

TABLE 15 ANTIBODY COMBINATIONS TESTED IN i-STAT ® CARTRIDGES Combination# ANA Beads Conjugate 1 5409 T25C 2 5409 10-1179-45 3 10-518-308 T25C 410-518-308 10-1179-45

These four sublots of cartridges (Table 15) were tested against a seriesof TSH in-house controls including stripped TSH plasma, at 0.1, 0.4,0.6, 1, 8, 45, 88 mIU/L levels for the sensitivity and linearityevaluation. They were also tested against spiked LH, FSH at 500 mIU/mland CG at 200000 mIU/ml in 0.4 mIU/L TSH samples for thecross-reactivity evaluation.

Based on the above tests, the TSH antibody clone 10-1179-456 wasconfirmed to be a potential candidate as replacing the Fitzgerald T25Cantibody to pair with the antibody Biospacific 5409 as this combinationhas similar signal response to TSH and less than 10% cross-reactivitywith LH, FSH and CG cross-reactants compared to the T25C/5409combination.

While the invention has been described in terms of various preferredembodiments, those skilled in the art will recognize that variousmodifications, substitutions, omissions and changes can be made withoutdeparting from the spirit of the present invention. Accordingly, it isintended that the scope of the present invention be limited solely bythe scope of the following claims.

What is claimed is:
 1. A thyroid stimulating hormone (TSH) sandwichimmunoassay, comprising at least two epitope-compatible antibodiescomprising at least one capture antibody and at least one signalantibody, wherein the dissociation constant (Kd) of both antibodies forTSH is between 0.08 and 0.1 nM, and wherein the capture antibody Kd forfollicle stimulating hormone (FSH), luteinizing hormone (LH) andchorionic gonadotropin (CG) is substantially 1000 nM, 1000 nM, and 500nM, respectively, and wherein the immunoassay uses a single wash step.2. An immunoassay of claim 1, wherein the signal antibody Kd for FSH, LHand CG is substantially 300 nM, 35 nM, and 40 nM, respectively.
 3. Animmunoassay of claim 1, wherein the at least one capture antibody ismouse monoclonal anti-TSH antibody
 5409. 4. An immunoassay of claim 1,wherein the at least one signal antibody is mouse monoclonal anti-TSHantibody M94206.
 5. An immunoassay of claim 1, wherein the at least onecapture antibody is mouse monoclonal anti-TSH antibody 5409 and the atleast one signal antibody is mouse monoclonal anti-TSH antibody M94206.6. An immunoassay of claim 1, wherein the at least one capture antibodyand the at least one signal antibody, respectively, bind to at least twodifferent epitopes on the TSH-β subunit of TSH.
 7. A thyroid stimulatinghormone (TSH) sandwich immunoassay, comprising at least twoepitope-compatible antibodies comprising at least one capture antibodyand at least one signal antibody, wherein the dissociation constant (Kd)of both antibodies for TSH is between 0.04 and 0.1 nM, and wherein thecapture antibody Kd for follicle stimulating hormone (FSH), luteinizinghormone (LH) and chorionic gonadotropin (CG) is substantially 1000 nM,1000 nM, and 500 nM, respectively, and wherein the immunoassay uses asingle wash step.
 8. An immunoassay of claim 7, wherein the signalantibody Kd for FSH, LH and CG is substantially 70 nM, 54 nM, and 45 nM,respectively.
 9. An immunoassay of claim 7, wherein the at least onecapture antibody is mouse monoclonal anti-TSH antibody
 5409. 10. Animmunoassay of claim 7, wherein the at least one signal antibody ismouse monoclonal anti-TSH antibody Ab9390.
 11. An immunoassay of claim7, wherein the at least one capture antibody is mouse monoclonalanti-TSH antibody 5409 and the at least one signal antibody is mousemonoclonal anti-TSH antibody Ab9390.
 12. An immunoassay of claim 7,wherein the at least one capture antibody and the at least one signalantibody, respectively, bind to at least two different epitopes on theTSH-β subunit of TSH.